The articles set forth in the Background of the Invention are each incorporated herein by reference.
Mammalian proteins, for example, those derived from clinical samples such as whole blood, serum, plasma, cerebrospinal fluid, and urine, are useful as indicators of a disease state or a bodily condition. The amount and type of these proteins in the sample can provide a wealth of information to the investigator.
For example, the protein components of serum include albumin, alpha-1 lipoprotein, alpha-2 macroglobulin, beta-1 lipoprotein and immunoglobuiins (including gammaglobulins). Albumin, the major protein of serum, is usually present in a concentration of between 4.0 and 5.0 g/dL. Decreased concentration of albumin can be indicative of renal disease; increased concentration of albumin is characteristic of dehydration. Elevations of alpha-1 lipoprotein can be indicative of chronic alcoholism or hyperestrogenism due to, e.g., pregnancy. Elevated levels of beta-1 lipoprotein can be indicative of increased cholesterol levels.
Mammalian proteins are charged proteins containing both cationic and anionic moieties. They thus lend themselves to analysis by capillary zone electrophoresis ("CZE"). CZE is a technique which permits rapid and efficient separations of charged substances. In general, CZE involves introduction of a sample into a capillary tube, i.e. a tube having an internal diameter of from about 10 to about 2000 microns and the application of an electric field to the tube. The electric potential of the field both pulls the sample through the tube and separates it into its constituent parts. i.e., each of the sample constituents has its own individual electrophoretic mobility; those having greater mobility travel through the capillary tube faster than those with slower mobility. As a result, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. An on-line detector can be used to continuously monitor the separation and provide data as to the various constituents based upon the discrete zones.
CZE can be generally separated into two categories based upon the contents of the capillary columns. In "gel" CZE, the capillary tube is filled with a suitable gel e.g., polyacrylamide gel. Separation of the constituents in the sample is predicated in part by the size and charge of the constituents travelling through the gel matrix. In "open" CZE, the capillary tube is filled with an electrically conductive buffer solution. Upon ionization of the capillary, the negatively charged capillary wall will attract a layer of positive ions from the buffer. As these ions flow towards the cathode, under the influence of the electrical potential, the bulk solution (i.e., the buffer solution and the sample being analyzed), must also flow in this direction to maintain electroneutrality. This electroendosmatic flow provides a fixed velocity component which drives both neutral species and ionic species, regardless of charge, towards the cathode. The buffer in open CZE is as stable against conduction and diffusion as the gels utilized in gel CZE. Accordingly, separations can be obtained in open CZE quite similar to those obtained in gel-based electrophoresis.
Fused silica is principally utilized as the material for the capillary tube because it can withstand the relatively high voltage used in CZE, and because the inner walls ionize to create the negative charge which causes the desired electroosmatic flow. The ionization of the inner walls of the capillary tube does, however, create problems with respect to separation of proteins.
This is because proteins are hetero-polyelectrolytes (i.e. an approximate equivalent number of positively and negatively charged moieties within the molecule when the molecule itself has a neutral-charge). Thus, when ionized, a protein species can have a net positive charge distribution such that the protein species will adsorb quite strongly onto the inner wall. This adsorption leads to artificial zone broadening in CZE, thus leading to inconclusive, erroneous or incomprehensible results.
One proposed attempt at solving this problem was to treat, or "coat", the inner wall of the capillary tube so that electroosmotic flow would be reduced when voltage was applied. That would, in turn, reduce adsorption of proteins onto the tube. Glycol modified fused silica capillaries have been used for serum protein analysis, but only with limited success. See Jorgenson, J. W. & Lukacs, K. D. "Capillary Zone Electrophoresis." Science 222:266-272 (1983). This is because treated fused silica capillaries have a relatively short shelf-life and their coatings have a tendency to "dissolve" in an unpredictable manner. The aura of unpredicitability is unacceptable in any environment where multiple samples will be analyzed on a frequent basis.
Another proposed solution to this problem was to use a buffer having a pH greater than the isoelectric points (pI) of the protein components of the sample. As is well known, when the pH is equal to the pI, the positive and negative moieties of the molecule are balanced. Similarly, when the pH is greater than pI, the negative moieties predominate and when the pH is less than the pI, the positive moieties exceed the negative moieties. For example, the pI of albumin is 4.6; therefore, at pH 4.6, the negatively charged and positively charged moieties of albumin are equal and the overall charge is neutral. However, as the pH is raised above the isoelectric point, the negatively charged moieties predominate and the net charge is negative. Thus, under the influence of a high pH buffer, all of the protein species of the sample will have a negative charge and will be repelled from the negatively charged wall. This will, in turn, avoid or at least greatly diminish their surface adsorption. However, large pH-pI differences can cause structural changes in the protein or even hydrolysis. Attempts to electrophorese proteins in untreated fused-silica capillary tubes using buffer solutions having pH ranges from 8-11 have resulted in irreproducible migration of all sample zones. See Lauer, H. H. and McManigill, D. "Capillary Zone Electrophoresis of Proteins in Untreated Fused Silica Tubing." Anal. Chem. 58:166-169 (1986).
It has been theorized that protein adsorption onto the untreated fused capillary wall is due to ion exchange interactions between cationic sites in the protein and silicate moieties in the wall. See Jorgenson, J. W. "Capillary Electrophoresis", Chpt. 13, New Direction in Electrophoretic Methods. ACS Symp. Ser. 335, 1987 (Jorgenson, J. W. & Phillips, M., Eds.). Accordingly, it has been suggested to use high salt buffer conditions to reduce protein adsorption. See Lauer, H. H. & McManigill, D., Trends Anal. Chem. 5:11 (1986). However, increasing the salt concentration of the buffer has the effect of increasing the conductivity of the capillary tube which can dramatically increase the heat inside the tube. Increasing temperature causes the migrating zones to become diffused, thus decreasing resolution of the zones. In order to avoid such heat build-up, the electric potential applied to the capillary tube must be greatly diminished. This, however, has the undesirable effect of increasing the time necessary for analysis of the sample.
Because capillary zone electrophoresis is such an extremely powerful tool for the separation of ionic species, a need exists for CZE analysis of protein and protein-portion ("peptide") samples which does not suffer from the severe and deleterious drawbacks noted above.